ABSTRACTOwing to the wide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life, toxicological research on ZnO NPs is receiving increasing attention in these days. In this study, the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cell line LTEP-a-2 was tested in vitro. Log-phase cells were exposed to different levels of ZnO NPs for hours, followed by colorimetric cell viability assay using tetrazolium salt and cell survival rate assay using trypan blue dye. Cell morphological changes were observed by Giemsa staining and light microscopy. Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay. Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method. Results showed that ZnO NPs (≥ 0.01 μg/mL) significantly inhibited proliferation (P < 0.05) and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure. The intracellular ROS level rose up to 30-40% corresponding to significant depletion (approximately 70-80%) in GSH content in LTEP-a-2 cells (P < 0.05), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production. This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells and provides reference data for application of nanomaterials in the environment.

Mentions:
The dye exclusion test was used to determine the number of viable cells present in a cell suspension. This method is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, eosin, or propidium, whereas dead cells do not [27]. In this test, a cell suspension was simply mixed with dye and then visually examined to determine whether cells take up or exclude dye. A viable cell was identified with a clear cytoplasm and a nonviable cell with a blue cytoplasm. Results showed that after 12 h of exposure to ZnO NPs (0.05–5.0 μg/mL), the survival rate of LTEP-a-2 cells underwent substantial decreases in a concentrate-dependent manner (Figure 3). In the presence of low concentration of ZnO NPs (0.05 μg/mL), cell survival rate remained above 60%, showing a nearly 40% decrease relative to the control treatment; as the concentration of ZnO NPs was increased to 0.1 μg/mL, cell survival rate underwent another 40% decrease, down to approximately 20% only. Together, these results confirm that the presence of ZnO NPs significantly affected cell survival even at low concentrations (e.g., 0.05–0.1 μg/mL).

Mentions:
The dye exclusion test was used to determine the number of viable cells present in a cell suspension. This method is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, eosin, or propidium, whereas dead cells do not [27]. In this test, a cell suspension was simply mixed with dye and then visually examined to determine whether cells take up or exclude dye. A viable cell was identified with a clear cytoplasm and a nonviable cell with a blue cytoplasm. Results showed that after 12 h of exposure to ZnO NPs (0.05–5.0 μg/mL), the survival rate of LTEP-a-2 cells underwent substantial decreases in a concentrate-dependent manner (Figure 3). In the presence of low concentration of ZnO NPs (0.05 μg/mL), cell survival rate remained above 60%, showing a nearly 40% decrease relative to the control treatment; as the concentration of ZnO NPs was increased to 0.1 μg/mL, cell survival rate underwent another 40% decrease, down to approximately 20% only. Together, these results confirm that the presence of ZnO NPs significantly affected cell survival even at low concentrations (e.g., 0.05–0.1 μg/mL).

ABSTRACTOwing to the wide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life, toxicological research on ZnO NPs is receiving increasing attention in these days. In this study, the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cell line LTEP-a-2 was tested in vitro. Log-phase cells were exposed to different levels of ZnO NPs for hours, followed by colorimetric cell viability assay using tetrazolium salt and cell survival rate assay using trypan blue dye. Cell morphological changes were observed by Giemsa staining and light microscopy. Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay. Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method. Results showed that ZnO NPs (≥ 0.01 μg/mL) significantly inhibited proliferation (P < 0.05) and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure. The intracellular ROS level rose up to 30-40% corresponding to significant depletion (approximately 70-80%) in GSH content in LTEP-a-2 cells (P < 0.05), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production. This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells and provides reference data for application of nanomaterials in the environment.